During hematopoiesis, pluripotent stem cells and their progeny undergo proliferation and differentiation, resulting in hematopoietic cells of various lineages. In some systems, decisions that determine the fate of stem cells and their progeny have been shown to result from intercellular interactions with adjacent cells, modulate by several families, including the Notch gene family. Our preliminary evidence supporting a role for the Notch receptor in regulating hematopoiesis includes the demonstration that 1) these receptors are widely expressed by hematopoietic cells, including hematopoietic precursor cells; and 2) differentiation of hematopoietic precursor cells in inhibited when they are induced to over- express the constitutively active cytoplasmic domain of Notch-1 or when they are exposed to exogenously presented Notch ligand forms. We propose to focus on the role of Notch signaling in the generation of stem cells with short- and long-term marrow repopulating ability. Specifically, we will evaluate the short- and long-term repopulating activity of cytokine-dependent, activated Notch-1-expressing hematopoietic precursor cells (Aim 1). Based on our data indicating different effects of Notch-1 versus Notch-2-induced signaling on the differentiation of 32D myeloid cells, we will investigate whether differing effects will result from over-expression of activated forms of other Notch homologs (Aim II). We will then determine whether ligand-induced Notch signaling has effects similar to those due to activated Notch in transduced cells by evaluating the effects of an immobilized form of the Notch ligand, Delta-1, on non-transduced hematopoietic precursors (Aim III). Since differential effects of Notch ligands have been observed in other cell systems, we will also determine whether ligand forms derived from other Notch ligands, including Jagged-1, Jagged-2, Delta-2, and Delta-3, enhance stem cell self-renewal more efficiently as compared to Delta-1 (Aim IV). Methods successful for enhancing marrow repopulating activity in mice will be evaluated in non-human primates in collaboration with Project VI (Aim V). These studies will provide insight into the role of this highly conserved receptor family in regulating mammalian human hematopoietic stem cell proliferation and differentiation in vitro.